Repertoire of antigen receptors (TCRs) on T cells is shaped by interactions between TCRs and MHC molecules bound with peptides of self and non-self origin. In thymus, these interactions promote differentiation of T cells with TCRs with low/intermediate affinity for self-MHC/peptide complexes. However, the dogma that only TCRs with low/intermediate affinity for self promote T cell differentiation has been challenged by the reports demonstrating that CD4+ regulatory cells often express high affinity, autoreactive TCRs. This unique feature of TCRs on regulatory CD4+ cells would direct these cells lineage commitment in the thymus and initiate expression of specific transcription factor Foxp3. Here we would like to examine if autoreactive TCRs are obligatory for regulatory T cells maturation and suppressive functions. Because natural diversity of TCRs on T cells is enormous, we have made mice where T cells are constrained to express the mini-repertoire of TCRs (TCRmini mice). In this model, individual T cells can be comprehensively monitored during their development in the thymus and later in the periphery. In TCRmini mice CD4+ T cells naturally mature as naive and regulatory and we further crossed these mice with mice expressing GFP reporter gene under Foxp3 regulatory sequences. Using these different strains of TCRmini mice we will examine if tissue specific peptides, which expression in the thymus is controlled by AIRE transcription factor, participate in conversion of CD4+Foxp3- medullary thymocytes to CD4+Foxp3+ lineage. These experiments will be performed using TCRmini AIRE+ and AIRE- mice. We also purpose to investigate the physiological relevance of presence of cognate antigen in the thymus on the differentiation of CD4+Foxp3+ T cells. We believe that these studies will reveal how TCR/MHC/peptide interactions govern the function and providence of Foxp3+ regulatory T cells in vivo. PUBLIC HEALTH RELEVANCE: This proposal focuses on investigating the mechanisms of maturation of T lymphocytes that naturally have suppressive and regulatory function. These regulatory T lymphocytes supervise the function of other immune cells. Better understanding how these cells differentiate is critical for the development of new strategies to treat autoimmune, infectious and neoplastic diseases.